RANJEET KUMAR | Örebro University (original) (raw)

Papers by RANJEET KUMAR

Understanding Tuberculosis - Deciphering the Secret Life of the Bacilli, 2012

Nucleic acids research, 2012

The ribosomal stalk in bacteria is composed of four or six copies of L12 proteins arranged in dim... more The ribosomal stalk in bacteria is composed of four or six copies of L12 proteins arranged in dimers that bind to the adjacent sites on protein L10, spanning 10 amino acids each from the L10 C-terminus. To study why multiple L12 dimers are required on the ribosome, we created a chromosomally engineered Escherichia coli strain, JE105, in which the peripheral L12 dimer binding site was deleted. Thus JE105 harbors ribosomes with only a single L12 dimer. Compared to MG1655, the parental strain with two L12 dimers, JE105 showed significant growth defect suggesting suboptimal function of the ribosomes with one L12 dimer. When tested in a cell-free reconstituted transcription-translation assay the synthesis of a full-length protein, firefly luciferase, was notably slower with JE105 70S ribosomes and 50S subunits. Further, in vitro analysis by fast kinetics revealed that single L12 dimer ribosomes from JE105 are defective in two major steps of translation, namely initiation and elongation involving translational GTPases IF2 and EF-G. Varying number of L12 dimers on the ribosome can be a mechanism in bacteria for modulating the rate of translation in response to growth condition.

European Biophysics Journal With Biophysics Letters, 2010

Malate synthase G is an important housekeeping enzyme of glyoxylate shunt in mycobacterium. The p... more Malate synthase G is an important housekeeping enzyme of glyoxylate shunt in mycobacterium. The pleotropic function of this protein by virtue of its intracellular/extracellular localization and its behavior as an adhesin and virulence factor is quite enigmatic. Despite its importance in mycobacterium persistence, we do not know much about its biophysical and biochemical properties. Earlier reports suggest that the enzyme exists only as a monomer in prokaryotes; however, we observed the existence of both active monomer and dimer forms of the enzyme under physiological conditions. The dimeric form of the enzymes is more stable as compared to the monomeric form as evident from various biophysical parameters. In addition, the dimeric enzyme also shows enhanced stability against proteolysis than the monomers. Based on these studies, it seems that dimerization is an important factor in regulating stability. The differential localization and diverse functions of malate synthase other than its enzymatic role might be triggering the stabilization of the enzyme dimer and modulation of activity and stability in vivo.

International Journal of Biological Macromolecules, 2011

Metabolic plasticity of Mycobacterium renders high degree of adaptive advantages in the persisten... more Metabolic plasticity of Mycobacterium renders high degree of adaptive advantages in the persistence through the upregulation of glyoxylate shunt. The malate synthase (MS), an important enzyme of the shunt belongs to the G isoform and expressed predominantly as monomer. Here we did a comparative unfolding studies of two homologous MS from Mycobacterium tuberculosis (MtbMS) and Escherichia coli (ecMS) using various biophysical techniques. Despite having high sequence identities, they show different structural, stability and functional properties. The study suggests that the differences in the stability and unfolding of the two enzymes are by virtue of differential electrostatic modulation unique to their respective molecular assembly.

Proteins-structure Function and Bioinformatics, 2008

Isocitrate lyase (Icl), an enzyme that plays an important role in the regulation of isocitrate fl... more Isocitrate lyase (Icl), an enzyme that plays an important role in the regulation of isocitrate flux and anaplerotic replenishment of pool of substrate required for biosynthetic process in Mycobacterium tuberculosis is a potential drug target for the antituberculosis drugs. Divalent cations induce differential effect of activation and inhibition of MtbIcl functional activity. The study for the first time demonstrates that interaction of cations with MtbIcl results in differential modulation of the enzyme structure which is probably the underlying mechanism for differential modulation of functional activity of enzyme by divalent cations. The Mg2+ and Mn2+ ions act as activators of the enzyme and in their absence no enzymatic activity was observed. These cations do not induce any significant structural alteration in the enzyme as observed by far-UV CD and solvent denaturation studies using chaotropic salts. However, the thermal denaturation studies demonstrate that they do interact with the noncatalytic α/β barrel core domain of the enzyme and destabilize it. The inhibitors Zn2+ and Cd2+ interact directly with the catalytic domain of the enzyme and unfold it as a result of which complete loss of the enzymatic activity is observed in their presence. The results obtained from the studies provide intriguing insight into the possible mechanism of divalent cation-induced changes in structure, function, and stability of MtbIcl. Proteins 2008. © 2008 Wiley-Liss, Inc.

Molecular and Cellular Endocrinology, 2005

The presence of estrogen receptor beta and aromatase in the germ cell has highlighted the physiol... more The presence of estrogen receptor beta and aromatase in the germ cell has highlighted the physiological role of the traditionally female hormone, estrogen, in spermatogenesis. Estrogen receptor alpha knockouts and aromatase knockouts have further accentuated the role of estrogen in germ cell maturation. To delineate the direct action of estrogen in the seminiferous epithelium, we studied the effects of high intratesticular estradiol. The study was based on the fact that administration of exogenous estradiol suppresses the hypothalamus pituitary gonadal axis (HPG) with a dose-dependant concomitant increase in intratesticular estrogen levels. Three doses of 17-β estradiol, namely 20, 100 and 200 μg/kg/day were administered subcutaneously to different batches of adult male rats for 10 days. The effect of the three doses on serum hormonal profile, intratesticular testosterone (T) and estradiol (E) levels were studied. Twenty micrograms per kilograms per day of 17-β estradiol affected the hypothalamus–pituitary axis, reducing serum gonadotropins and intratesticular testosterone; however, 100 μg/kg/day of 17-β estradiol decreased serum FSH and intratesticular testosterone, increased intratesticular estradiol, but had no effect on serum LH. Interestingly, 200 μg/kg/day of 17-β estradiol decreased serum and intratesticular T without any effect on serum gonadotropins. This could be attributed to the positive feedback effect of estrogens on gonadotropins. In the testis, morphologically two visible effects were seen, namely ‘spermiation failure’ in all three doses attributed to the suppression of T and FSH and a ‘maintenance effect’ in the 100 μg/kg/day attributed to E and/or 10% of available intratesticular T. The direct effect of an increase in intratesticular estradiol levels was observed in terms of a decrease in apoptosis in germ cell. The study, therefore, suggests that 100 μg/kg/day of 17-β estradiol could be used to study the effects of high intratesticular estradiol with a concomitant decrease in intratesticular T and serum FSH levels on spermatogenesis.

Understanding Tuberculosis - Deciphering the Secret Life of the Bacilli, 2012

Nucleic acids research, 2012

The ribosomal stalk in bacteria is composed of four or six copies of L12 proteins arranged in dim... more The ribosomal stalk in bacteria is composed of four or six copies of L12 proteins arranged in dimers that bind to the adjacent sites on protein L10, spanning 10 amino acids each from the L10 C-terminus. To study why multiple L12 dimers are required on the ribosome, we created a chromosomally engineered Escherichia coli strain, JE105, in which the peripheral L12 dimer binding site was deleted. Thus JE105 harbors ribosomes with only a single L12 dimer. Compared to MG1655, the parental strain with two L12 dimers, JE105 showed significant growth defect suggesting suboptimal function of the ribosomes with one L12 dimer. When tested in a cell-free reconstituted transcription-translation assay the synthesis of a full-length protein, firefly luciferase, was notably slower with JE105 70S ribosomes and 50S subunits. Further, in vitro analysis by fast kinetics revealed that single L12 dimer ribosomes from JE105 are defective in two major steps of translation, namely initiation and elongation involving translational GTPases IF2 and EF-G. Varying number of L12 dimers on the ribosome can be a mechanism in bacteria for modulating the rate of translation in response to growth condition.

European Biophysics Journal With Biophysics Letters, 2010

Malate synthase G is an important housekeeping enzyme of glyoxylate shunt in mycobacterium. The p... more Malate synthase G is an important housekeeping enzyme of glyoxylate shunt in mycobacterium. The pleotropic function of this protein by virtue of its intracellular/extracellular localization and its behavior as an adhesin and virulence factor is quite enigmatic. Despite its importance in mycobacterium persistence, we do not know much about its biophysical and biochemical properties. Earlier reports suggest that the enzyme exists only as a monomer in prokaryotes; however, we observed the existence of both active monomer and dimer forms of the enzyme under physiological conditions. The dimeric form of the enzymes is more stable as compared to the monomeric form as evident from various biophysical parameters. In addition, the dimeric enzyme also shows enhanced stability against proteolysis than the monomers. Based on these studies, it seems that dimerization is an important factor in regulating stability. The differential localization and diverse functions of malate synthase other than its enzymatic role might be triggering the stabilization of the enzyme dimer and modulation of activity and stability in vivo.

International Journal of Biological Macromolecules, 2011

Metabolic plasticity of Mycobacterium renders high degree of adaptive advantages in the persisten... more Metabolic plasticity of Mycobacterium renders high degree of adaptive advantages in the persistence through the upregulation of glyoxylate shunt. The malate synthase (MS), an important enzyme of the shunt belongs to the G isoform and expressed predominantly as monomer. Here we did a comparative unfolding studies of two homologous MS from Mycobacterium tuberculosis (MtbMS) and Escherichia coli (ecMS) using various biophysical techniques. Despite having high sequence identities, they show different structural, stability and functional properties. The study suggests that the differences in the stability and unfolding of the two enzymes are by virtue of differential electrostatic modulation unique to their respective molecular assembly.

Proteins-structure Function and Bioinformatics, 2008

Isocitrate lyase (Icl), an enzyme that plays an important role in the regulation of isocitrate fl... more Isocitrate lyase (Icl), an enzyme that plays an important role in the regulation of isocitrate flux and anaplerotic replenishment of pool of substrate required for biosynthetic process in Mycobacterium tuberculosis is a potential drug target for the antituberculosis drugs. Divalent cations induce differential effect of activation and inhibition of MtbIcl functional activity. The study for the first time demonstrates that interaction of cations with MtbIcl results in differential modulation of the enzyme structure which is probably the underlying mechanism for differential modulation of functional activity of enzyme by divalent cations. The Mg2+ and Mn2+ ions act as activators of the enzyme and in their absence no enzymatic activity was observed. These cations do not induce any significant structural alteration in the enzyme as observed by far-UV CD and solvent denaturation studies using chaotropic salts. However, the thermal denaturation studies demonstrate that they do interact with the noncatalytic α/β barrel core domain of the enzyme and destabilize it. The inhibitors Zn2+ and Cd2+ interact directly with the catalytic domain of the enzyme and unfold it as a result of which complete loss of the enzymatic activity is observed in their presence. The results obtained from the studies provide intriguing insight into the possible mechanism of divalent cation-induced changes in structure, function, and stability of MtbIcl. Proteins 2008. © 2008 Wiley-Liss, Inc.

Molecular and Cellular Endocrinology, 2005

The presence of estrogen receptor beta and aromatase in the germ cell has highlighted the physiol... more The presence of estrogen receptor beta and aromatase in the germ cell has highlighted the physiological role of the traditionally female hormone, estrogen, in spermatogenesis. Estrogen receptor alpha knockouts and aromatase knockouts have further accentuated the role of estrogen in germ cell maturation. To delineate the direct action of estrogen in the seminiferous epithelium, we studied the effects of high intratesticular estradiol. The study was based on the fact that administration of exogenous estradiol suppresses the hypothalamus pituitary gonadal axis (HPG) with a dose-dependant concomitant increase in intratesticular estrogen levels. Three doses of 17-β estradiol, namely 20, 100 and 200 μg/kg/day were administered subcutaneously to different batches of adult male rats for 10 days. The effect of the three doses on serum hormonal profile, intratesticular testosterone (T) and estradiol (E) levels were studied. Twenty micrograms per kilograms per day of 17-β estradiol affected the hypothalamus–pituitary axis, reducing serum gonadotropins and intratesticular testosterone; however, 100 μg/kg/day of 17-β estradiol decreased serum FSH and intratesticular testosterone, increased intratesticular estradiol, but had no effect on serum LH. Interestingly, 200 μg/kg/day of 17-β estradiol decreased serum and intratesticular T without any effect on serum gonadotropins. This could be attributed to the positive feedback effect of estrogens on gonadotropins. In the testis, morphologically two visible effects were seen, namely ‘spermiation failure’ in all three doses attributed to the suppression of T and FSH and a ‘maintenance effect’ in the 100 μg/kg/day attributed to E and/or 10% of available intratesticular T. The direct effect of an increase in intratesticular estradiol levels was observed in terms of a decrease in apoptosis in germ cell. The study, therefore, suggests that 100 μg/kg/day of 17-β estradiol could be used to study the effects of high intratesticular estradiol with a concomitant decrease in intratesticular T and serum FSH levels on spermatogenesis.